1. Field of the Invention
The present invention relates to novel calcium phosphate core particles, to methods of making them, and to methods of using them as vaccine adjuvants, as cores or carriers for biologically active material, and as controlled release matrices for biologically active material.
2. Description of Related Art
Nanometer scale particles have been proposed for use as carrier particles, as supports for biologically active molecules, such as proteins, and as decoy viruses. See U.S. Pat. Nos. 5,178,882; 5,219,577; 5,306,508; 5,334,394; 5,460,830; 5,460,831; 5,462,750; and 5,464,634, the entire contents of each of which are hereby incorporated by reference.
The particles disclosed in the above-referenced patents, however, are generally extremely small, in the 10-200 nm size range. Particles of this size are difficult to make with any degree of consistency, and their morphology is not described in any detail. None of these patents disclose the use of nanoparticles as sustained release matrices. Furthermore, these patents do not disclose the use of calcium phosphate particles as either (1) adjuvants for vaccines or viral decoys, or (2) controlled release matrices for delivery of pharmaceuticals or immunogenic materials.
There has been a suggestion in the literature to use calcium phosphate particles as vaccine adjuvants, but calcium phosphate particles have generally been considered an unsuitable alternative to other adjuvants due to inferior adjuvanting activity. See, e.g., Goto et al., Vaccine, vol. 15, No. 12/13 (1997). Moreover, the calcium phosphate evaluated was typically microparticulate (>1000 nm diameter) and possessed a rough and oblong morphology, in contrast to the core particles of the present invention.
Therefore, an important need remains for calcium phosphate core particles useful as core materials or carriers for biologically active moieties which can be produced simply and consistently. A further need remains for calcium phosphate core particles that can be effectively, used as adjuvants for vaccines, as cores or carriers for biologically active molecules, and as controlled release matrices.
There is also a need for calcium phosphate core particles that can be effectively used as supports and matrices for sustained release of polynucleotide material (DNA or RNA) encoding immunogenic polypeptides. Traditional vaccination involves exposing a potential host to attenuated or killed pathogens, or immunogenic components thereof (e.g., proteins or glycoproteins). The basic strategy has changed little since the development of the first smallpox vaccine nearly a century ago, although modern developments permit genetic engineering of recombinant protein vaccines. However, traditional vaccine methodologies may be undesirable as a result of their expense, instability, poor immunogenicity, limited heterogeneity and potential infectivity.
Polynucleotide vaccination presents a different vaccine methodology, whereby polynucleotide material, such as DNA or RNA, encoding an immunogenic polypeptide is delivered intracellularly to a potential host. The genetic material is taken up and expressed by these cells, leading to both a humoral and a cell-mediated immune response. It is not entirely clear whether DNA vaccines function as a result of integration or simply long-term episomal maintenance.
Polynucleotide vaccination provides numerous advantages over traditional vaccination. Polynucleotide vaccines eliminate the risk of infection associated with live attenuated viruses, yet advantageously induce both humoral and cell-mediated responses. Polynucleotide vaccines further provide prolonged immunogen expression, generating significant immunological memory and eliminating the need for multiple inoculations. Polynucleotide vaccines are very stable, permitting prolonged storage, transport and distribution under variable conditions. As a further advantage, a single polynucleotide vaccine may be engineered to provide multiple immunogenic polypeptides. Thus, a single DNA vaccine can be used to immunize against multiple pathogens, or multiple strains of the same pathogen. Finally, polynucleotide vaccines are much simpler and less expensive to manufacture than traditional vaccines.
Polynucleotide vaccines may take various forms. The genetic material can be provided, for example, in combination with adjuvants capable of stimulating the immune response. Administration of the DNA or RNA coated onto microscopic beads has been suggested. See J. J. Donnelly et al., Annu. Rev. Immunol. 15, 617 (1997). Various routes of administration are also possible, and may include, for example, intravenous, subcutaneous and intramuscular administration.
A desirable immune response to an immunogenic polypeptide is two-fold, involving both humoral and cellular-mediated immunity. The humoral component involves stimulation of B cells to product antibodies capable of recognizing extracellular pathogens, while the cell-mediated component involves T lymphocytes capable of recognizing intracellular pathogens. Cytotoxic T-lymphocytes (CTLs) play an important role in the latter, by lysing virally-infected or bacterially-infected cells. Specifically, CTLs possess receptors capable of recognizing foreign peptides associated with MHC class I and/or class II molecules. These peptides can be derived from endogenously synthesized foreign proteins, regardless of the protein's location or function within the pathogen. Thus, CTLs can recognize epitopes derived from conserved internal viral proteins (J. W. Yewdell et al., Proc. Natl. Acad. Sci. (USA) 82, 1785 (1985); A. R. M. Towsend, et al., Cell 44, 959 (1986); A. J., McMichael et al., J. Gen. Virol. 67, 719 (1986); A. R. M. Towsend and H., Annu. Rev. Immunol. 7, 601 (1989)) and may therefore permit heterologous protection against viruses with multiple serotypes or high mutation rates. Polynucleotide vaccination can stimulate both forms of immune response, and thus is very desirable.
Efforts to use polynucleotide vaccination have focused on the use of viral vectors to deliver polynucleotides to host cells. J. R. Bennink et al., 311, 578 (1984); J. R. Bennink and J. W. Yewdell, Curr. Top. Microbiol. Immunol. 163, 153 (1990); C. K. Stover et al., Nature 351, 456 (1991); A. Aldovini and R. A. Young, Nature 351, 479 (1991); R. Schafer et al., J. Immunol. 149, 53 (1992); C. S. Hahn et al., Proc. Nail. Acad. Sci. (USA) 89, 2679 (1992). However, this approach may be undesirable for several reasons. Retroviral vectors, for example, have restrictions on the size and structure of polypeptides that can be expressed as fusion proteins while maintaining the ability of the recombinant virus to replicate (A. D. Miller, Curr. Top. Microbiol. Immunol. 158, 1 (1992). The effectiveness of vectors such as vaccinia for subsequent immunizations may be compromised by immune responses against vaccinia (E. L. Cooney et al., Lancet 337, 567 (1991)). Also, viral vectors and modified pathogens have inherent risks that may hinder their use in humans (R. R. Redfield et al., New Engl. J. Med. 316, 673 (1987); L. Mascola et al., Arch. Intern. Med. 149, 1569 (1989)). For example, in live vector approaches, highly immunogenic vectors also tend to be highly pathogenic.
Alternative gene delivery methods have also been explored. Benvenisty, N., and Reshef, L. (PNAS 83, 9551-9555, (1986)) showed that CaCl2 precipitated DNA could be expressed in mice. Plasmid vectors have also been used to produce expression in mouse muscle cells (J. A. Wolff et al., Science 247, 1465 (1990); G. Ascadi et al., Nature 352, 815 (1991)). The plasmids were shown to be maintained episomally and did not replicate. Subsequently, persistent expression has been observed after i.m. injection in skeletal muscle of rats, fish and primates, and cardiac muscle of rats (H. Lin et al., Circulation 82, 2217 (1990); R. N. Kitsis et al., Proc. Natl. Acad. Sci. (USA) 88, 4138 (1991); E. Hansen et al., FEBS Lett. 290, 73 (1991); S. Jiao et al., Hum. Gene Therapy 3, 21 (1992); J. A. Wolff et al., Human Mol. Genet. 1, 363 (1992)). WO 90/11092 (4 Oct. 1990) reported the use of naked polynucleotides to vaccinate vertebrates.
Various routes of administration have been found to be suitable for vaccination using polynucleotide vaccines. Intramuscular administration is thought to be particularly desirable, given the proportionally large muscle mass and its direct'accessibility through the skin. See U.S. Pat. No. 5,580,859. Tang et al., (Nature, 356, 152-154 (1992)) disclosed that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice. Furth et al., (Analytical Biochemistry, 205, 365-368, (1992)) showed that a jet injector could be used to transfect skin, muscle, fat, and mammary tissues of living animals. WO 93/17706 describes a vaccination method wherein carrier particles are coated with a gene construct and then accelerated into a potential host. Intravenous injection of a DNA:cationic liposome complex in mice has also been reported (Zhu et al., Science 261, 209-211 (9 Jul. 1993); see also WO 93/24640). Methods for introducing nucleic acids have been reviewed (Friedman, T., Science, 244, 1275-1281 (1989)); see also Robinson et al., (Abstracts of Papers Presented at the 1992 meeting on Modern Approaches to New Vaccines, Including Prevention of AIDS, Cold Spring Harbor, p 92; Vaccine 11, 957 (1993)), where the intra-muscular, intra-venous, and intra-peritoneal administration of avian influenza DNA into chickens was alleged to have provided protection against lethal challenge.
Reports suggest that polynucleotide vaccination has provided effective protective immunity in various animal models. The immunization of mice against influenza by the injection of plasmids encoding influenza A hemagglutinin has been reported (Montgomery, D. L. et al., 1993, Cell Biol., 12, pp. 777-783), or nucleoprotein (Montgomery, D. L. et at, supra; Ulmer, J. B. et al., 1993, Science, 259, pp. 1745-4749). The first use of DNA immunization for a herpes virus has been reported (Cox et al., 1993, J. Viral., 67, pp. 5664-5667). Injection of a plasmid encoding bovine herpes virus 1 (BHV-1) glycoprotein g IV gave rise to anti-g IV antibodies in mice and calves. Upon intranasal challenge with BHV-1, immunized calves showed reduced symptoms and shed substantially less virus than controls. Wang et al., (P.N.A.S. USA 90, 4156-4160 (May 1993)) reported on elicitation of immune responses in mice against HIV by intramuscular inoculation with a cloned, genomic (unspliced) HIV gene. However, the level of immune responses achieved was very low, and the system utilized portions of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter and portions of the simian virus 40 (SV40) promoter and terminator. SV40 is known to transform cells, possibly through integration into host cellular DNA. Thus, the system described by Wang et al., may be inappropriate for administration to humans.
It has been suggested to use calcium phosphate particles as agents for transfection of therapeutic polynucleotides in gene therapy. See U.S. Pat. No. 5,460,831. DNA or RNA is attached to the particulate core and delivered to a target cell, resulting in expression of therapeutic proteins. However, this patent does not suggest the use of calcium phosphate particles as supports for DNA or RNA vaccines. To the contrary, this patent indicates that the stimulation of an immunological response during transfection is to be avoided. This patent also fails to suggest the use of calcium phosphate particles as controlled release matrices for genetic material.
There is also a need for calcium phosphate core particles that can be used effectively used as an inhalable aerosol delivery system for the delivery of therapeutic proteins or peptide agents, and in particular, for delivery of insulin and other hormones. For a number of therapeutic agents, delivery of the agent to a patient in need thereof can be difficult. This is particularly true with proteins and peptides, which are difficult or impossible to administer orally, since they are easily digested or hydrolyzed by the enzymes and other components of gastric juices and other fluids secreted by the digestive tract. Injection is often the primary alternative administration method, but is unpleasant, expensive and is not well tolerated by patients requiring treatment for chronic illnesses. In particular, patients who are administered drugs on an out-patient basis, or who self-administer, are more likely to fail to comply with the required administration schedule. A particular group of patients of this type are those suffering from diabetes, who frequently must inject themselves with insulin in order to maintain appropriate blood glucose levels.
Recently, alternative methods of administration therapeutic agents have been sought, in particular, administration by inhalation of an aerosol containing the therapeutic agent. The lungs can be used effectively to get the therapeutic agent into the bloodstream because they have a very large surface area of very thin tissue. As a result, for some therapeutic agents and delivery systems, the level of agent in the blood can rise as fast as, or faster than, that obtained when the agent is administered by injection beneath the skin. Moreover, the thin lung tissue allows the passage of proteins and peptides into the blood stream without exposing them to the type or level of proteases encountered during oral administration.
Aerosols containing the therapeutic agent as fine, suspended mists of particles in both liquid and solid form have been investigated. However, preparation of suitable inhalable aerosols can be difficult for therapeutic agents where the blood level of the agent is critical, e.g., with insulin, because the amount of aerosol delivered to the deep lung tissue can be substantially variable, leading to inconsistent dosages of the drug to the patient.
As a result of this need to provide a reliable inhalable aerosol delivery system, various attempts have been made to develop small, solid particles for the delivery of therapeutic agents via inhalation. For example, an inhalable form of insulin is reportedly under development wherein the insulin is combined with sugar particles of a particular size to make an ultrafine powder that is delivered when it is forced through an inhaler nozzle by a blast of compressed air. See R. F. Service, Science 277:5330 (1997). Another inhalable form of insulin involves relatively large (diameters>5 μm), porous polymer particles (50:50 poly(lactic acid-co-glycolic acid) of low density (ρ<˜0.4 g/cm3) that encapsulate insulin. The particles are believed to penetrate deep into the lung tissue as the result of their low density, yet avoid phagocytosis when in the tissue as the result of their large size. See D. E. Edwards et al., Science, 276:1868 (1997).
Despite these attempts, there remains a need for an inhalable aerosol delivery system that effectively provides consistent, reliable, therapeutic blood levels of protein or peptide therapeutic agents, and in particular, of insulin and other hormones. It is particularly desirable that any carrier material be very small and easily biodegradable, in order to avoid complications resulting from inhalation of particulates.